Cytosolic free calcium elevation mediates the phagosome-lysosome fusion during phagocytosis in human neutrophils.

Cytosolic free calcium ([Ca2+]i) and fusion of secondary granules with the phagosomal membrane (phagosome-lysosome fusion, P-L fusion) were assessed in single adherent human neutrophils during phagocytosis of C3bi-opsonized yeast particles. Neutrophils were loaded with the fluorescent dye fura2/AM and [Ca2+]i was assessed by dual excitation microfluorimetry. Discharge of lactoferrin, a secondary granule marker into the phagosome was verified by immunostaining using standard epifluorescence, confocal laser scanning and electron microscopy. In Ca2(+)-containing medium, upon contact with a yeast particle, a rapid rise in [Ca2+]i was observed, followed by one or more Ca2+ peaks (maximal value 1,586 nM and median duration 145 s): P-L fusion was detected in 80% of the cells after 5-10 min. In Ca2(+)-free medium the amplitude, frequency and duration of the [Ca2+]i transients were decreased (maximal value 368 nM, mostly one single Ca2+ peak and median duration 75 s): P-L fusion was decreased to 52%. Increasing the cytosolic Ca2+ buffering capacity by loading the cells with MAPT/AM led to a dose-dependent inhibition both of [Ca2+]i elevations and P-L fusion. Under conditions where basal [Ca2+]i was reduced to less than 20 nM and intracellular Ca2+ stores were depleted, P-L fusion was drastically inhibited while the cells ingested yeast particles normally. P-L fusion could be restored in Ca2(+)-buffered cells containing ingested particles by elevating [Ca2+]i with the Ca2(+)-ionophore ionomycin. The present findings directly indicate that although the ingestion step of phagocytosis is a Ca2(+)-independent event, [Ca2+]i transients triggered upon contact with opsonized particles are necessary to control the subsequent fusion of secondary granules with the phagosomal membrane

Gene products of chromosome 11q and their association with CCND1 gene amplification and tamoxifen resistance in premenopausal breast cancer

Introduction: The amplification event occurring at chromosome locus 11q13, reported in several different cancers, includes a number of potential oncogenes. We have previously reported amplification of one such oncogene, namely CCND1, to be correlated with an adverse effect of tamoxifen in premenopausal breast cancer patients. Over-expression of cyclin D-1 protein, however, confers tamoxifen resistance but not a tamoxifen-induced adverse effect. Potentially, co-amplification of an additional 11q13 gene, with a resulting protein over-expression, is required to cause an agonistic effect. Moreover, during 11q13 amplification a deletion of the distal 11q region has been described. In order to assess the potential impact of the deletion we examined a selected marker for this event. Method: Array comparative genomic hybridization analysis was employed to identify and confirm changes in the gene expression of a number of different genes mapping to the 11q chromosomal region, associated with CCND1 amplification. The subsequent protein expression of these candidate genes was then examined in a clinical material of 500 primary breast cancers from premenopausal patients who were randomly assigned to either tamoxifen or no adjuvant treatment. The protein expression was also compared with gene expression data in a subset of 56 breast cancer samples. Results: Cortactin and FADD (Fas-associated death domain) over-expression was linked to CCND1 amplification, determined by fluorescence in situ hybridization, but was not associated with a diminished effect of tamoxifen. However, deletion of distal chromosome 11q, defined as downregulation of the marker Chk1 (checkpoint kinase 1), was associated with an impaired tamoxifen response, and interestingly with low proliferative breast cancer of low grade. For Pak1 (p21-activated kinase 1) and cyclin D-1 the protein expression corresponded to the gene expression data. Conclusions: The results indicate that many 11q13 associated gene products are over-expressed in conjunction with cyclin D-1 but not linked to an agonistic effect of tamoxifen. Finally, the deletion of distal 11q, linked to 11q13 amplification, might be an important event affecting breast cancer outcome and tamoxifen response

Simulation-based design study for the passive shielding of the COSINUS dark matter experiment

The COSINUS (Cryogenic Observatory for SIgnatures seen in Next-generation Underground Searches) experiment aims at the detection of dark matter-induced recoils in sodium iodide (NaI) crystals operated as scintillating cryogenic calorimeters. The detection of both scintillation light and phonons allows performing an event-by-event signal to background discrimination, thus enhancing the sensitivity of the experiment. The construction of the experimental facility is foreseen to start by 2021 at the INFN Gran Sasso National Laboratory (LNGS) in Italy. It consists of a cryostat housing the target crystals shielded from the external radioactivity by a water tank acting, at the same time, as an active veto against cosmic ray-induced events. Taking into account both environmental radioactivity and intrinsic contamination of materials used for cryostat, shielding and infrastructure, we performed a careful background budget estimation. The goal is to evaluate the number of events that could mimic or interfere with signal detection while optimising the geometry of the experimental setup. In this paper we present the results of the detailed Monte Carlo simulations we performed, together with the final design of the setup that minimises the residual amount of background particles reaching the detector volume.Comment: 12 pages, 7 figure

Time-dynamic effects on the global temperature when harvesting logging residues for bioenergy

The climate mitigation potential of using logging residues (tree tops and branches) for bioenergy has been debated. In this study, a time-dependent life cycle assessment (LCA) was performed using a single-stand perspective. Three forest stands located in different Swedish climate zones were studied in order to assess the global temperature change when using logging residues for producing district heating. These systems were compared with two fossil reference systems in which the logging residues were assumed to remain in the forest to decompose over time, while coal or natural gas was used for energy. The results showed that replacing coal with logging residues gave a direct climate benefit from a single-stand perspective, while replacing natural gas gave a delayed climate benefit of around 8-12 years depending on climate zone. A sensitivity analysis showed that the time was strongly dependent on the assumptions for extraction and combustion of natural gas. The LCA showed that from a single-stand perspective, harvesting logging residues for bioenergy in the south of Sweden would give the highest temperature change mitigation potential per energy unit. However, the differences between the three climate zones studied per energy unit were relatively small. On a hectare basis, the southern forest stand would generate more biomass compared to the central and northern locations, which thereby could replace more fossil fuel and give larger climate benefits

Deep-underground dark matter search with a COSINUS detector prototype

Sodium iodide (NaI) based cryogenic scintillating calorimeters using quantum sensors for signal read out have shown promising first results towards a model-independent test of the annually modulating signal detected by the DAMA/LIBRA dark matter experiment. The COSINUS collaboration has previously reported on the first above-ground measurements using a dual channel readout of phonons and light based on transition edge sensors (TESs) that allows for particle discrimination on an event-by-event basis. In this letter, we outline the first underground measurement of a NaI cryogenic calorimeter read out via the novel remoTES scheme. A 3.67 g NaI absorber with an improved silicon light detector design was operated at the Laboratori Nazionali del Gran Sasso, Italy. A significant improvement in the discrimination power of $e^-$/$\gamma$-events to nuclear recoils was observed with a five-fold improvement in the nuclear recoil baseline resolution, achieving $\sigma$ = 441 eV. Furthermore, we present a limit on the spin-independent dark-matter nucleon elastic scattering cross-section achieving a sensitivity of $\mathcal{O}$(pb) with an exposure of only 11.6 g d.Comment: 11 pages, 14 figure

Весовые соотношения тимуса и надпочечников у антенатально погибших плодов

ТИМУСНАДПОЧЕЧНИКИтимомегалиядети первого года жизнидети раннего возрастаМЛАДЕНЕЦ, СМЕРТНОСТЬЭНДОКРИННОЙ СИСТЕМЫ БОЛЕЗНИСМЕРТНОСТЬ ДЕТСКАЯсиндром внезапной детской смерт

Resistance to First-Line Anti-TB Drugs Is Associated with Reduced Nitric Oxide Susceptibility in Mycobacterium tuberculosis

Background and objective: The relative contribution of nitric oxide (NO) to the killing of Mycobacterium tuberculosis in human tuberculosis (TB) is controversial, although this has been firmly established in rodents. Studies have demonstrated that clinical strains of M. tuberculosis differ in susceptibility to NO, but how this correlates to drug resistance and clinical outcome is not known. Methods: In this study, 50 sputum smear- and culture-positive patients with pulmonary TB in Gondar, Ethiopia were included. Clinical parameters were recorded and drug susceptibility profile and spoligotyping patterns were investigated. NO susceptibility was studied by exposing the strains to the NO donor DETA/NO. Results: Clinical isolates of M. tuberculosis showed a dose- and time-dependent response when exposed to NO. The most frequent spoligotypes found were CAS1-Delhi and T3_ETH in a total of nine known spoligotypes and four orphan patterns. There was a significant association between reduced susceptibility to NO (>10% survival after exposure to 1mM DETA/NO) and resistance against first-line anti-TB drugs, in particular isoniazid (INH). Patients infected with strains of M. tuberculosis with reduced susceptibility to NO showed no difference in cure rate or other clinical parameters, but a tendency towards lower rate of weight gain after two months of treatment. Conclusion: There is a correlation between resistance to first-line anti-TB drugs and reduced NO susceptibility in clinical strains of M. tuberculosis. Further studies including the mechanisms of reduced NO susceptibility are warranted and could identify targets for new therapeutic interventions

Reliability of cyclin A assessment on tissue microarrays in breast cancer compared to conventional histological slides

Cyclin A has in some studies been associated with poor breast cancer survival, although all studies have not confirmed this. Its prognostic significance in breast cancer needs evaluation in larger studies. Tissue microarray (TMA) technique allows a simultaneous analysis of large amount of tumours on a single microscopic slide. This makes a rapid screening of molecular markers in large amount of tumours possible. Because only a small tissue sample of each tumour is punched on an array, the question has arisen about the representativeness of TMA when studying markers that are expressed in only a small proportion of cells. For this reason, we wanted to compare cyclin A expression on TMA and on traditional large sections. Two breast cancer TMAs were constructed of 200 breast tumours diagnosed between 1997–1998. TMA slides and traditional large section slides of these 200 tumours were stained with cyclin A antibody and analysed by two independent readers. The reproducibility of the two readers' results was good or even very good, with kappa values 0.71–0.87. The agreement of TMA and large section results was good with kappa value 0.62–0.75. Cyclin A overexpression was significantly (P<0.001) associated with oestrogen receptor and progesterone receptor negativity and high grade both on TMA and large sections. Cyclin A overexpression was significantly associated with poor metastasis-free survival both on TMA and large sections. The relative risks for metastasis were similar on TMA and large sections. This study suggests that TMA technique could be useful to study histological correlations and prognostic significance of cyclin A on breast cancer on a large scale

Democratic research: Setting up a research commons for a qualitative, comparative, longitudinal interview study during the COVID-19 pandemic

The sudden and dramatic advent of the COVID-19 pandemic led to urgent demands for timely, relevant, yet rigorous research. This paper discusses the origin, design, and execution of the SolPan research commons, a large-scale, international, comparative, qualitative research project that sought to respond to the need for knowledge among researchers and policymakers in times of crisis. The form of organization as a research commons is characterized by an underlying solidaristic attitude of its members and its intrinsic organizational features in which research data and knowledge in the study is shared and jointly owned. As such, the project is peer-governed, rooted in (idealist) social values of academia, and aims at providing tools and benefits for its members. In this paper, we discuss challenges and solutions for qualitative studies that seek to operate as research commons

Human Macrophages Infected with a High Burden of ESAT-6-Expressing M. tuberculosis Undergo Caspase-1- and Cathepsin B-Independent Necrosis

Mycobacterium tuberculosis (Mtb) infects lung macrophages, which instead of killing the pathogen can be manipulated by the bacilli, creating an environment suitable for intracellular replication and spread to adjacent cells. The role of host cell death during Mtb infection is debated because the bacilli have been shown to be both anti-apoptotic, keeping the host cell alive to avoid the antimicrobial effects of apoptosis, and pro-necrotic, killing the host macrophage to allow infection of neighboring cells. Since mycobacteria activate the NLRP3 inflammasome in macrophages, we investigated whether Mtb could induce one of the recently described inflammasome-linked cell death modes pyroptosis and pyronecrosis. These are mediated through caspase-1 and cathepsin-B, respectively. Human monocyte-derived macrophages were infected with virulent (H37Rv) Mtb at a multiplicity of infection (MOI) of 1 or 10. The higher MOI resulted in strongly enhanced release of IL-1β, while a low MOI gave no IL-1β response. The infected macrophages were collected and cell viability in terms of the integrity of DNA, mitochondria and the plasma membrane was determined. We found that infection with H37Rv at MOI 10, but not MOI 1, over two days led to extensive DNA fragmentation, loss of mitochondrial membrane potential, loss of plasma membrane integrity, and HMGB1 release. Although we observed plasma membrane permeabilization and IL-1β release from infected cells, the cell death induced by Mtb was not dependent on caspase-1 or cathepsin B. It was, however, dependent on mycobacterial expression of ESAT-6. We conclude that as virulent Mtb reaches a threshold number of bacilli inside the human macrophage, ESAT-6-dependent necrosis occurs, activating caspase-1 in the process